Abstract

Background: Recently vivax severity has been on the rise in India where P. vivax contributes in equal ratio with P. falciparum to the malaria incidence. Objectives:We report here two cases of vivax malaria- one severe and another non-severe case, diagnosed and confirmed by microscopy, rapid diagnostic tests and 18S rRNA PCR assay.

Methods: Quantitative expression of two drug resistance genes (pvcrt-o, pvmdr1) and five vir (variant interspersed repeats) genes were measured simultaneously in these two cases for evaluating their role in disease pathogenesis. The non-severe case was taken as a control for measuring the expression levels of the studied genes in the severe case.

Results: The results indicated that clinical severe case was due to P.vivax only. The transcript levels of pvcrt-o and pvmdr1 alongwith the four vir genes were seen to be significantly high when compared to the non-severe vivax malaria case in the study.

Keywords

Severe vivax, Cerebral malaria, Drug resistance, vir genes.

Introduction

P. vivax is the most widespread species of human malaria
across the world transmitted in 95 countries where an
estimated 2.85 billion people are at risk of infection [1].
Although P. falciparum is responsible for majority of
severe manifestations of malaria and mortality associated
with it worldwide, P. vivax is no more considered benign
as it is emerging to be equally complicated and lethal [2].
Many studies from India [3,4] as well as other parts of the
world like Indonesian Papua [5,6], Papua New Guinea [7]
and Brazilian Amazon [8,9] show a marked correlation
between vivax infections, severe disease and death.

Previous studies have shown major clinical manifestations
exclusively linked to severe P. vivax which include
cerebral malaria, renal failure, circulatory collapse, ARDS,
jaundice, severe anemia, thrombocytopenia, multiorgan
dysfunction (MOD), possible coma which could clinically
lead to life-threatening episodes [2,10]. Cerebral malaria
and severe thrombocytopenia are reported as frequent
clinical complications associated with vivax malaria,
which were earlier exclusive for P. falciparum [11-13].
Parasitaemias in vivax infections are generally low and
severe disease is not characterized by high parasite load
[14]. Reports from different P. vivax endemic regions
suggest the range and rate of severe clinical complications associated to P. vivax to be very diverse that could be due
to many unexplained factors linked to the parasite, host
and the type of infection [15].

Not much information is available on the genes that could
have a role to play in the complicated vivax malaria.
The vir (variant interspersed repeats) genes, the largest
subtelomeric multigene superfamily found in P. vivax belonging to variant surface antigen (VSA) family, is
proposed to have a role in the antigenic variation of the
parasite and these genes are found to be highly variant
[16,17]. Five vir genes were chosen for analysis in the
present study according to their in silico data and a previous
understanding of their speculated role in the pathogenesis
of P. vivax [16,18,19].

Even though chloroquine resistance (CQR) in P. falciparum is found to be associated to mutations in pfcrt and pfmdr1 genes, the orthologous genes in P. vivax, P. vivax chloroquine resistance transporter (pvcrt-o) and
the P. vivax multidrug resistance transporter (pvmdr1)
are not yet considered as genetic markers for CQR in P. vivax [20,21]. However previous studies have shown
significant increase in the expression levels of these genes
in clinically severe vivax infections as well as chloroquine
resistant cases suggesting a possible association of CQR
and P. vivax severity [22,23]. The variant vir genes, pvcrt-o and pvmdr1 genes need to be explored as possible genetic
markers for disease severity to gain a better understanding
of the clinical and epidemiological mechanisms.

Case study

Patient 1

A 14-year old female was brought in and admitted to
Kalawati Saran Children’s Hospital, a tertiary care
hospital in New Delhi, in September 2013, with repeated
convulsions since the past two days, giddiness, skin
rash, muscle aches and recurring fever with chills. On
arrival, the patient was febrile (38.3°C), confused and
unresponsive. Family members revealed no prior history
of convulsions. Vital signs included a blood pressure of
100/75 mm of Hg, 20 breaths per minute and a pulse
rate of 110 beats per minute. Chest examinations were
normal and spleen was palpable 3 cm below the costal
margin. Laboratory investigations included the following
results: hemoglobin = 6.8 g/dL (normal range:11.5-15.5
g/dL); platelets= 7100/μl (normal range:150,000-400,000
μl); total bilirubin = 2.9 mg/dL (normal range: 0.3- 1.9
mg/dL); serum creatinine = 1.2 mg/dL (normal range:
0.1- 1.0 mg/dL); total leukocyte count was 4900/μl with
30% neutrophils, 50% lymphocytes and 18% monocytes
and serum electrolytes were within normal limits. Blood
cultures and biochemical tests for other co-morbidities like
serology against hepatitis A, hepatitis E, HIV, leptospirosis
and dengue were performed and were found to be negative.
Absence of other co-morbidities like pneumonia, enteric
fever, varicella-zoster virus, diabetes, hypertension etc.
was also confirmed. Lumbar puncture and CT scans were
performed without any pathological findings.

Microscopic slides of thick and thin Giemsa-stained blood
smears showed the presence of trophozoites and schizonts
of P. vivax with 2.0% parasitaemia. Rapid diagnostic
tests (RDTs) (FalciVax Zephyr Biomedical systems) also
confirmed the presence of P. vivax in the blood sample.
Two ml venous blood was collected from the patient and
about 50 μl of the venous blood that was collected was
used to make filter paper blood spots on Whatman filter
paper (number 3) for the parasite genomic DNA extraction
from QIAamp DNA Blood Mini Kit (Qiagen Inc.)
according to the manufacturer’s instructions. A further
diagnostic confirmation of P. vivax mono-infection was
made by 18S rRNA nested PCR assay to amplify speciesspecfic
sequences of the small subunit of rRNA genes of
P.falciparum, P.vivax and P.malariae and also confirmed
the absence of P. falciparum and P.malariae co-infection
[24].

The tests confirmed that the patient was suffering from
cerebral malaria and severe thrombocytopenia solely due
to P. vivax. Patient was treated with injection ceftriaxone
and artesunate in combination with primaquine along
with intravenous (IV) fluids. Platelet transfusion was also
administered. The patient made full recovery in six days.

Patient 2

A single uncomplicated vivax malaria case was used as
control in this study who was treated in the same hospital
and was discharged after all the tests were carried out. The
uncomplicated P. vivax malaria patient was a 12-year old
male who was brought in with recurring fever with chills
(38°C) since the past three days and headache. Laboratory
investigations showed all results within the normal range
and preliminary diagnosis with RDT and microscopy
revealed the presence of P. vivax infection. Thick and thin
Geimsa-stained blood smears showed P. vivax asexual
stages, trophozoites and schizonts with 1.5% parasitaemia.
Diagnostic confirmation of Plasmodium species was done
by 18S rRNA PCR assay which corroborated the results
of microscopy and RDT showing P. vivax monoinfection
in the patient. This uncomplicated vivax malaria patient
was treated with chloroquine (25 mg/kg) for three days
following which, the patient recovered.

The infected blood from both patients was passed through
CF-11 column to remove the leukocytes. P. vivax total
RNA was isolated by the QIAamp RNA Blood Mini Kit
(Qiagen Inc.) according to the manufacturer’s instructions.
First strand cDNA was then synthesized from 150 ng of
total RNA using oligo (dT)18 primers (Thermo Scientific)
according to the manufacturer’s protocol.

Relative quantification by real-time PCR was carried out to
find the expression levels of five vir genes (vir 14-related, vir 12, vir 17-like, putative vir 14 and vir 10-related)
and P. vivax drug resistance genes (pvcrt-o and pvmdr1).
β-tubulin was used as the endogenous gene in this study
as it has been used in similar studies previously [23].
Primers for pvcrt-o, pvmdr1, vir genes and β-tubulin were
designed by Primer3web (v 4.0.0) software to compare
the transcript levels of these genes [22]. The severe vivax
isolate was normalized against the control uncomplicated
vivax isolate by 2-ΔΔCt method for relative quantification of
the drug resistance and vir genes.

It was found that the expression levels of pvcrt-o, pvmdr1 and four out of five vir genes (vir 14-related, vir 12, vir
17-like and vir 10-related) were several fold higher in the
severe vivax isolate as compared to the uncomplicated
control isolate. The highest expression was seen in vir 10-related gene followed by vir 12, vir 17-like, vir
14-related, pvmdr1 and pvcrt-o genes (Figure 1). Putative vir 14 gene was not expressed in the test and control
isolates.

Discussion

The severe case of P. vivax reported here confirms the emerging severity due to vivax malaria in the country.
Several reports from different endemic regions reveal
diverse range and rate of occurrence of severe P. vivax
clinical cases, which was earlier limited to P. falciparum infections only [2,5,6,8,9]. Cerebral malaria has been
mainly associated with P. falciparum infections and very
few such cases due to vivax have been reported only
recently from several regions of the country and from
other countries too [2,3,11,25,26].

Previous studies have elaborated that severe manifestations
for malaria like cerebral malaria, hepatic dysfunction,
acute renal failure and ARDS are caused by the
sequestration of infected erythrocytes in deep vasculature
of vital organs whereas thrombocytopenia and severe
anemia are caused by factors like hemolysis, reduced cell
deformability of infected and uninfected erythrocytes,
increased splenic clearance, increased splenic uptake of
platelets and decreased platelet production and survival
[27,28]. Our patient exhibited cerebral malaria and
severe thrombocytopenia exclusively due to P. vivax
malaria as stated previously in the manuscript. Previous
data indicates that P. vivax can cause both sequestration
and non-sequestration related clinical manifestations
[2]. In several studies, severe anemia has been the most
frequently associated manifestation in severe P. vivax
[2,6,8,13]. Also, it has been observed that the occurrence
of thrombocytopenia in P. vivax malaria is on the rise
[12,29,30]. During the preliminary examination of a
febrile patient, if thrombocytopenia is also observed it
should be borne in mind to get the patient investigated for
malaria infection. The occurrence of two or more severity
criteria is now frequently observed in severe vivax
cases [31]. Similarly in our severe case, we observed
two severe clinical manifestations i.e. convulsions and
thrombocytopenia which have also been seen elsewhere
[2,11-13,25,26]. It was not possible in our study to estimate
the total parasite biomass which was a limitation in our
study though, in the severe malaria criteria bilirubin and low hemoglobin levels are important diagnostic markers
and should not be excluded in the initial diagnosis of P.
vivax infected patients.

The expression levels of the drug resistance genes viz. pvcrt-o and pvmdr1 possibly having a role in chloroquine
resistance and disease severity was seen to be significantly
high when compared to the non-severe vivax malaria
case in the study. The pvcrt-o gene expression was 11.16
fold higher and pvmdr1 gene expression was 12.37 fold
higher in severe vivax case normalized to the non-severe
infection.

Species specific 18S rRNA PCR technique confirmed
that P. vivax alone was responsible for the severe
manifestations of the patient under study. Many studies
have been conducted in the past from Papua Indonesia,
Manaus Brazil, Venezuela and India that have highlighted
the occurrence of severe P. vivax infections leading to
life threatening episodes more in children as compared to
adults in regions of high vivax transmission [6,9,32,33].
Lanca et al., observed severe anemia and respiratory
distress to be the most frequent complications in their
study.

We require more comprehensive studies to elucidate the
role of drug resistance and virulence genes in severe
vivax infections. The molecular mechanisms need to
be unraveled for detailed understanding of the disease
pathogenesis of vivax malaria, which is now severe like P. falciparum. The malaria burden due to P. vivax needs to be
examined urgently for implementation of adequate control
measures in the national control program.

Acknowledgements

This work was supported by the Indian Council of Medical
Research [F/801/2010-ECD-II, ICMR/5/8-7(247)/V-
2012-ECD-II]. We would also like to acknowledge the
patients included in the study.